Method for quickly detecting bifidobacterium based on high-throughput sequencing and application

A bifidobacteria and gene technology, applied in the field of molecular biology, can solve the problems of low resolution, affecting the industrial application of bifidobacteria, and restricting the research of bifidobacteria

Active Publication Date: 2018-02-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The high-throughput sequencing technology based on 16S rRNA gene has the characteristics of simplicity and rapidity, which makes it possible to detect bifidobacteria in the intestinal tract. Low limitations, so this method can not meet the detection of Bifidobacterium species and subspecies level
This limits the research of bifidobacteria to a certain extent, thereby affecting the industrial application of bifidobacteria

Method used

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  • Method for quickly detecting bifidobacterium based on high-throughput sequencing and application
  • Method for quickly detecting bifidobacterium based on high-throughput sequencing and application
  • Method for quickly detecting bifidobacterium based on high-throughput sequencing and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Follow the steps below:

[0038] 1. By downloading 114 groEL gene sequences of different species of Bifidobacterium, construct a phylogenetic tree through MEGA software, such as figure 1 As shown, we can see from the figure that this gene can well distinguish different species of Bifidobacteria, so this gene can be used for identification and detection of different species of Bifidobacteria.

[0039] 2. Construct a groEL gene database, download known groEL gene sequences of bifidobacteria from databases such as NCBI (National Center for Biotechnology Information) and EMBL (European Molecular Biology Laboratory), and use the downloaded sequences to construct a groEL gene comparison database. The constructed groEL gene sequence database is applicable to all known bifidobacteria.

[0040] 3. Adopt the method of high-throughput sequencing to collect the DNA information of the microbial genome of the complex sample to be tested; the extraction of the microbial genome refers...

Embodiment 2

[0045] The detection of different kinds of bifidobacteria in the human feces sample of embodiment 2

[0046] 1. Take a human feces sample, and extract the genomic DNA of the microorganisms in the feces sample, using the DNA extraction kit from MPBiomedicals, USA. The specific operation steps are subject to the operation manual of the kit.

[0047] 2. Design and synthesize Bifidobacterium-specific primer sequences

[0048] Forward primer (SEQ ID NO.1): 5'-TCCGATTACGAYCGYGAGAAGCT-3'

[0049] Reverse primer (SEQ ID NO.2): 5'-CSGCYTCGGTSGTCAGGAACAG-3'

[0050] 3. Use the above-mentioned forward primer and reverse primer, and use the extracted genomic DNA as a template to carry out PCR amplification. The PCR amplification reaction system consists of: genomic DNA template 2ul, Ex taq mix 25ul (TaKaRa), 20uM Each lul of forward primer and reverse primer, add ddH 2 0 to 50ul.

[0051] The reaction conditions of the PCR amplification were: pre-denaturation at 95°C for 5 min, follo...

Embodiment 3

[0055] Determination of content of different species of bifidobacteria in embodiment 3 human feces samples

[0056] 1. According to the method described in steps 1-4 in Example 2, the only difference is that the primer is a general primer for the V3-V4 region of 16S rRNA, which amplifies the V3-V4 region of all microorganisms in human feces, and the database of the V3-V4 region obtained by sequencing The percentage of Bifidobacterium in the total bacterial content was obtained. The specific results are shown in Table 1.

[0057] Table 1 The percentage of Bifidobacteria in the total bacteria in human feces samples

[0058]

[0059] 2. Multiply the percentage of each Bifidobacterium obtained in Step 5 in Example 2 by the content of Bifidobacteria in the stool sample in Step 1 to obtain the percentage of each Bifidobacterium species in human feces . The specific results are shown in Table 2.

[0060] Different species of bifidobacteria account for the percentage of total b...

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Abstract

The invention discloses a method for quickly detecting a bifidobacterium based on high-throughput sequencing and application, and belongs to the field of molecular biology. According to the method, agroEL gene is used as a screening marker; the high-throughput identification can be realized; the information of a strain can be processed in a batching manner; the separation and the purification arenot needed, so that components of the bifidobacterium in a complicated sample can be fully identified; moreover, a subspecies level can be reached; the recognition rate is higher than 16SrRNA (ribosomal Ribonucleic Acid). Through the specific-primer extension based on the groEL gene, and in comparison with a conventional 16SrRNA method, the identification period of the level of a bifidobacteriumspecies is at least shortened for the time of 1 day; the method provided by the invention can be used for identifying the bifidobacterium absolutely and the accuracy rates of the identification on a species and a subspecies reach 100 percent.

Description

technical field [0001] The invention relates to a high-throughput sequencing-based rapid detection method for bifidobacteria and its application, belonging to the technical field of molecular biology. Background technique [0002] Bifidobacteria, as important probiotics in the intestinal tract of humans and some other animals, have attracted the attention of experts and scholars in industry and academia. Bifidobacteria can not only inhibit the growth of pathogenic bacteria in the intestinal tract to regulate and improve the intestinal flora, but also can improve lactose intolerance, lower serum cholesterol, promote immunity and play anti-oxidation and other important beneficial effects. A large number of microecological studies at home and abroad have shown that bifidobacteria also have various functions such as antibacterial, anti-cancer, and nutritional. Therefore, it is particularly important to study bifidobacteria in the intestines of humans and other animals. However...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6869C12Q1/689C12Q1/6851C12Q1/04C12Q1/06
CPCC12Q1/6851C12Q1/6869C12Q1/689C12Q2535/122C12Q2531/113
Inventor 陈卫陆文伟胡陆军翟齐啸赵建新张灏
Owner JIANGNAN UNIV
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